Bioactive proteins are essential for the function of cells of living organisms, and are responsible for most of the activities of the cell including catalysis of metabolic processes, communication, defense, movement, and transport. Oral delivery of bioactive proteins is typically required for two principal purposes: delivery of therapeutic proteins and delivery of proteins that have nutritional or another beneficial effect on animals, including humans.
The growing demand for food over the world, particularly for animal protein, together with the awareness towards environmental impacts of animal growth, requires the development of sophisticated agricultural management tools that would improve the productivity and weight gain of terrestrial as well as aquatic farm animals. Development of aquacultures for the growth of marine animals including fish, crustaceans and mollusks is of particular importance, as marine animal are considered as an healthier source for proteins.
However, aquaculture is still not a completely efficient system for growing marine animals and fish in particular. Typically, fish require a relatively long period of time to reach an acceptable size and weight. Furthermore, many breeds of fish mature in an inefficient manner, such that problems may occur in the fish population, resulting in loss of a portion of the population due to excessively slow growth, poor morphology (for ornamental fish) and so forth. Furthermore, in the artificial aquaculture environment, the aquatic animals are more susceptible to infectious diseases and, when disease does occur, it can spread rapidly through entire populations with high mortality.
There is thus a need for routes to administer therapeutic or nutritional proteins to terrestrial as well as aquatic farm animals that are not costly and do not require laborious efforts. Oral delivery of therapeutic or nutritional proteins to humans is also highly desirable, as this mode of administration does not require professional manpower and significantly increases the patient compliance to the prescribed dose. The biological activity of a protein depends on its sequence and/or conformation, which must be preserved until the protein reaches its target of activity. For successful oral delivery, proteins should be protected from chemical and enzymatic degradation that may occur during processing the proteins into food or a feed composition and through the delivery via the animal's gastrointestinal tract. In addition, the protein should overcome structural barriers that preclude entry into the animal or access to the target destination.
Microalgae (single cell alga or phytoplankton) represent the largest, but most poorly understood, kingdom of microorganisms on the earth. Like plants are to terrestrial animals, the microalgae represent the natural nutritional base and primary source of all the phytonutrients in the aquatic food chain. Expression of recombinant proteins in algae has been reported, and various methods are available for production of exogenous proteins within the algae cells, particularly within the cell plastid. International (PCT) Application Publication No. WO 2011/063284 discloses methods of expressing therapeutic proteins in photosynthetic organisms, including prokaryotes such as cyanobacteria and eukaryotes such as alga and plants. Transformation of eukaryotes is preferably into the plastid genome, typically into the chloroplast genome. The Application discloses expression of particular therapeutic proteins within algae cells.
Various attempts have been made to use microalgae as delivery means for proteins. For example, International (PCT) Application Publication No. WO 01/98335 discloses delivery systems and methods for delivering a biologically active protein to a host animal. The systems and methods provided include obtaining an algal cell transformed by an expression vector, the expression vector comprising a nucleotide sequence coding for the biologically active protein, operably linked to a promoter. In one illustrated embodiment, the biologically active protein is an antigenic epitope and upon administration to the animal the algal cell induces an immune response in the host animal.
International (PCT) Application Publication No. WO 2002/076391 discloses the use of microbial cells which are used as feed components in aquaculture or agriculture, and which also contain exogenous peptides, proteins, and/or antibodies, which will convey resistance or immunity to viral or bacterial pathogens or otherwise improve the health and performance of the species consuming said microbial cells. The microbial cells can be yeast, fungi, bacteria, or algae. The proteins and/or antibodies may be expressed inside the microbial cells by direct genetic modification of the microbe itself, or by the infection of the microbe with a virus that has been altered to express the protein of interest.
International (PCT) Application Publication No. WO 2008/027235 discloses methods for prevention, amelioration or treatment of a disease or disorder in an aquatic animal, by feeding the aquatic animal directly or indirectly with genetically modified micro algae that expresses a recombinant molecule that specifically targets one or more key epitopes of a pathogen that infects the aquatic animal.
U.S. Patent Application Publication No. 2011/0014708 discloses method of producing a foreign desired gene product in algae that comprises weakening or removing the algae cell wall by a protein enzyme solution to facilitate the gene transfer and a feed composition comprising the transgenic algae or its offspring. The invention also provides a modified nucleic acid for expressing bovine lactoferricin (LFB) in algae.
However, there is still an unmeet need for and it would be highly advantageous to have an oral delivery system that is easy for production and use, maintains the biological activity of the protein and facilitate absorption of the biologically active protein systemically.